The long term goal of this research is to gain a deeper understanding of the structure, catalytic mechanism, and metabolic control of flavoproteins involved in fatty acid oxidation. Three proteins form the core of this proposal: the mitochondrial medium chain acyl-CoA dehydrogenase and electron-transferring flavoprotein (ETF), and the peroxisomal acyl-CoA oxidase. The molecular basis for chain length discrimination in the medium chain dehydrogenase will be studied by steady state and rapid reaction methods, using both normal substrates and a variety of redox-inactive acyl-CoA analogues. An unusual mode of oxidative inactivation of the dehydrogenase by the ferricenium ion will be studied, and the mechanism-based inhibition of the enzyme by 3,4-allenic thioesters will be .characterized. The mechanism of the inactivation of acyl-CoA oxidase by 2-bromo-hexadecanoyl-CoA and 2-hexadecynoyl-CoA will be studied, and the targets of these irreversible inhibitors established. The factors which control differences in acceptor specificity between the evolutionarily related acyl-CoA dehydrogenase and acyl-CoA oxidase will be explored. TRP166, which protects part of the si-face of the flavin ring in the medium chain dehydrogenase from solvent, will be replaced by other aromatic and aliphatic side chains to examine their effect on the reactivity of the mutant enzymes towards both the natural electron acceptor, ETF, and the non-physiological oxidant, molecular oxygen. Significant differences in acceptor specificity will be considered in terms of current ideas concerning the mechanism of biological electron transfer reactions and the modulation of oxygen reactivity in flavoproteins. Attempts will be made to identify and to explore the role of an apparently new prosthetic group found recently in mammalian ETF. A study of the Escherichia coli and Neurospora crassa acyl-CoA dehydrogenase and ETF systems will be initiated to see whether they offer significant advantages, or additional insight, concerning the mechanism of interflavin electron transfer between these redox partners.